Trichoderma spp. have been known as biocontrol agents since the 1930s (Weindling, R., “Trichoderma lignorum as a Parasite of Other Soil Fungi,” Phytopathology 22:837-845 (1932)) and have been shown to have dramatic effects on plants (Chet, I., “Innovative Approaches to Plant Disease Control,” In R. Mitchell (ed.), Wiley Series in Ecological and Applied Microbiology pp. 372. Jon Wiley & Sons, New York (1987); Harman, G., “Myths and Dogmas of Biocontrol. Changes in Perceptions Derived from Research on Trichoderma harzianum T-22,” Plant Dis. 84:377-393 (2000)). The effects noted include (a) increased growth and yields of plants (Chang et al., “Increased Growth of Plants in the Presence of the Biological Control Agent Trichoderma harzianum,” Plant Dis. 70:145-148 (1986); Harman, G., “Myths and Dogmas of Biocontrol. Changes in Perceptions Derived from Research on Trichoderma harzianum T-22,” Plant Dis. 84:377-393 (2000); Lindsey et al., “Effect of Certain Fungi on Dwarf Tomatoes Grown under Gnotobiotic Conditions,” Phytopathology 57:1262-1263 (1967); Yedidia et al., “Effect of Trichoderma harzianum on Microelement Concentrations and Increased Growth of Cucumber Plants,” Plant Soil 235:235-242 (2001)); (b) increased root growth and drought tolerance (Harman, G., “Myths and Dogmas of Biocontrol. Changes in Perceptions Derived from Research on Trichoderma harzianum T-22,” Plant Dis. 84:377-393 (2000)); (c) induced systemic resistance to disease (Geremia et al., “Molecular Characterization of the Proteinase-Encoding Gene, Prb1, Related to Mycoparasitism by Trichoderma harzianum,” Molec. Microbiol. 8:603-613 (1993); Harman et al., “Trichoderma Species—Opportunistic, Avirulent Plant Symbionts,” Nature Rev. Microbiol. 2:43-56 (2004); Yedidia et al., “Induction of Defense Responses in Cucumber Plants (Cucumis sativus L.) by the Biocontrol Agent Trichoderma harzianum,” Appl. Environ. Microbiol. 65:1061-1070 (1999); Yedidia et al., “Induction and Accumulation of PR Proteins Activity During Early Stages of Root Colonization by the Mycoparasite Trichoderma harzianum Strain T-203,” Plant Physiol. Biochem. 38:863-873 (2000); Yedidia et al., “Concomitant Induction of Systemic Resistance to Pseudomonas syringae pv. Lachrymans in Cucumber by Trichoderma asperellum (T-203) and Accumulation of Phytoalexins,” Appl. Environ. Microbiol. 69:7343-7353 (2003)); (d) increased nutrient uptake and fertilizer utilization efficiency (Harman, G., “Myths and Dogmas of Biocontrol. Changes in Perceptions Derived from Research on Trichoderma harzianum T-22,” Plant Dis. 84:377-393 (2000); Yedidia et al., “Effect of Trichoderma harzianum on Microelement Concentrations and Increased Growth of Cucumber Plants,” Plant Soil 235:235-242 (2001)); (e) increased leaf greenness, increased expression of proteins involved in photosynthesis and greater starch accumulation that is indicative of increased photosynthetic rate (Harman, G., “Myths and Dogmas of Biocontrol. Changes in Perceptions Derived from Research on Trichoderma harzianum T-22,” Plant Dis. 84:377-393 (2000); Harman et al., “The Mechanisms and Applications of Opportunistic Plant Symbionts,” In M. Vurro and J. Gressel (eds.), Novel Biotechnologies for Biocontrol Agent Enhancement and Management pp. 131-153. Springer, Amsterdam (2007)); and (f) increased percentages of germination and rates of germination of seeds (Bjorkman et al., “Growth Enhancement of Shrunken-2 Sweet Corn with Trichoderma harzianum 1295-22: Effect of Environmental Stress,” J. Am. Soc. Hort. Sci. 123:35-40 (1998); Chang et al., “Increased Growth of Plants in the Presence of the Biological Control Agent Trichoderma harzianum,” Plant Dis. 70:145-148 (1986)). In addition, Trichoderma strains alleviated effects of salt stress on squash plant growth (Yildirim et al., “Ameliorative Effects of Biological Treatments on the Growth of Squash Plants Under Salt Stress,” Sci. Hortic. 111:1-6 (2006)) and can overcome the negative effects of low levels of osmotic stress on germination of tomato seeds. Trichoderma strains can alleviate not only stresses extrinsic to plants, but also intrinsic stresses. Seeds lose vigor as they age but seed treatments with Trichoderma spp. can restore vigor and improve germination, even in the presence of any pathogenic organisms (Bjorkman et al., “Growth Enhancement of Shrunken-2 Sweet Corn with Trichoderma harzianum 1295-22: Effect of Environmental Stress,” J. Am. Soc. Hort. Sci. 123:35-40 (1998)).
This long list of effects indicates that the Trichoderma-plant interactions are complex. They must involve alterations in a wide range of plant metabolic pathways and, almost by definition, widespread changes in gene expression and in the physiology of plants. This indicates that Trichoderma can essentially re-program plant genes and protein expression, and generally this results in benefits to plant growth and productivity and in resistance to biotic and abiotic stresses, including those that occur intrinsically, such as via seed aging. This capability of a fungus to re-program a maize plant is not without precedent—the maize smut pathogen also has this ability (Doehlemann et al., “Reprogramming a Maize Plant: Transcriptional and Metabolic Changes Induced by the Fungal Biotroph Ustilago maydis,” Plant J. 56:181-95 (2008)).
Since 2000, the international Trichoderma research community, especially with the availability of “-omics” tools, has been able to arrive at a consensus as to the events that occur in the Trichoderma-plant interaction. Harman et al., “Trichoderma Species—Opportunistic, Avirulent Plant Symbionts,” Nature Rev. Microbiol. 2:43-56 (2004)) provides a complete review of such interactions. These Trichoderma-plant interactions can be summarized as follows: (a) Trichoderma strains colonize and infect the outer layers of roots (Yedidia et al., “Induction of Defense Responses in Cucumber Plants (Cucumis sativus L.) by the Biocontrol Agent Trichoderma harzianum,” Appl. Environ. Microbiol. 65:1061-1070 (1999); Yedidia et al., “Induction and Accumulation of PR Proteins Activity During Early Stages of Root Colonization by the Mycoparasite Trichoderma harzianum Strain T-203,” Plant Physiol. Biochem. 38:863-873 (2000)); (b) once infection occurs, a zone of chemical interaction develops at these sites. Within this zone of chemical interaction, the Trichoderma hyphae are walled off by the plant but are not killed (Harman et al., “Trichoderma Species—Opportunistic, Avirulent Plant Symbionts,” Nature Rev. Microbiol. 2:43-56 (2004); Harman et al., “The Mechanisms and Applications of Opportunistic Plant Symbionts,” In M. Vurro and J. Gressel (eds.), Novel Biotechnologies for Biocontrol Agent Enhancement and Management pp. 131-153. Springer, Amsterdam (2007)). This walling off is accomplished through the interaction of chemical elicitors from Trichoderma with plant receptors. Some of the elicitors now are known and the hypothesis that Trichoderma spp. induce wide-scale changes in the physiology of the plant holds true and has been verified by both proteomic and transcriptomic assays (Alfano et al., “Systemic Modulation of Gene Expression in Tomato by Trichoderma harzianum 382,” Phytopathology 97:429-437 (2007); Bailey et al., “Fungal and Plant Gene Expression During the Colonization of Cacao Seedlings by Endophytic Isolates of Four Trichoderma Species,” Planta (Berlin) 224:1449-1464 (2006); Djonovic et al., “Sm1, a Proteinaceous Elicitor Secreted by the Biocontrol Fungus Trichoderma virens Induces Plant Defense Responses and Systemic Resistance,” Molec. Plant Microbe Interact. 8:838-853 (2006); Djonovic et al., “A Proteinaceous Elicitor Sm1 from the Beneficial Fungus Trichoderma virens is Required for Systemic Resistance in Maize,” Plant Physiol. 145:875-889 (2007); Marra et al., “Biocontrol Interactions Involving Plants, Fungal Pathogens and Antagonists of the Genus Trichoderma,” Abstracts, XIII International Congress on Molecular Plant-Microbe Interactions: 399 (2007); Segarra et al., “Proteome, Salicylic Acid and Jasmonic Acid Changes in Cucumber Plants Inoculated with Trichoderma asperellum Strain T34,” Proteomics 7:3943-3952 (2007); Shoresh et al., “Characterization of a Mitogen-Activated Protein Kinase Gene from Cucumber Required for Trichoderma-Conferred Plant Resistance,” Plant Physiol. 142:1169-1179 (2006); Shoresh et al., “Genome-Wide Identification, Expression and Chromosomal Location of the Chitinase Genes in Zea mays,” Molec. Gen. Genom. 280:173-85 (2008); Viterbo et al., “The 18mer Peptaibols from Trichoderma virens Elicit Plant Defence Responses,” Molec. Plant Pathol. 8:737-746 (2007); Yedidia et al., “Induction and Accumulation of PR Proteins Activity During Early Stages of Root Colonization by the Mycoparasite Trichoderma harzianum Strain T-203,” Plant Physiol. Biochem. 38:863-873 (2000); Yedidia et al., “Concomitant Induction of Systemic Resistance to Pseudomonas syringae pv. Lachrymans in Cucumber by Trichoderma asperellum (T-203) and Accumulation of Phytoalexins,” Appl. Environ. Microbiol. 69:7343-7353 (2003)).
While the references cited represent major steps forward in the understanding of plant-Trichoderma interactions, it is just a beginning in an effort to elucidate the mechanisms and systems involved in this important plant-microbe symbiosis. An important fact is that strains differ substantially in their effects on plants. Only a few effectively enhance plant growth, induce high levels of systemic disease or stress resistance and provide the other advantages noted above. In large part, this is probably due to the fact that different strains produce different elicitors. Changes in gene expression can be used to formulate hypotheses regarding physiological changes in plants. For example, based on proteomic data, it can be hypothesized that T. harzianum strain T22 increases photosynthetic rates, respiration rates, and induces resistance to biotic and abiotic stresses.
It is worth noting that, while T22 has been widely used, other nonrhizosphere competent strains have largely fallen by the wayside, such as the strain described in Lumsden et al., “Isolation and Localization of the Antibiotic Gliotoxin Produced by Gliocladium virens from Alginate Prill in Soil and Soilless Media,” Phytopathology 82:230-235 (1992). This strain produces the antibiotic gliotoxin and protects plants for a few weeks. After that time, this strain, which is not rhizosphere competent, becomes quiescent and its activity is lost.
The interaction of Trichoderma strains with plants may increase the nitrogen use efficiency. Globally the nitrogen (N) problem is a big one. About 60 percent of streams sampled in the U.S. show some signs of excess nitrogen loading. The World Water Council, an independent association of water scientists and engineers, reported that more than half of the world's biggest fresh-water lakes are threatened by pollution or drainage schemes. Coastal ecosystems are also affected. The Gulf of Mexico, for example, contains a notorious oxygen-depleted “dead zone” caused by agricultural run-off from the Mississippi river. One study on the Mississippi River basin found that reducing fertilizer use by just 12 percent would reduce nitrogen runoff by 33 percent (McIsaac et al., “Nitrate Flux in the Mississippi River,” Nature 414:166-167 (2001)). This may occur because anthropogenic nitrogen application exceeds the capacity of terrestrial or aquatic systems to assimilate nitrogen input.
Further, three gases associated with agriculture—nitrous oxide (N2O), methane, and carbon dioxide—contribute to the level of greenhouse gases that are largely responsible for global warming. N2O is released from soils and its greenhouse warming potential (GWP) is 296 times greater than that of CO2 (Snyder, C., “Fertilizer Nitrogen BMPs to Limit Losses that Contribute to Global Warming,” International Plant Nutrition Institute, Norcross, Ga. (2008)). Best Management Practices can reduce N2O emissions by proper placement and application of nitrogen fertilizer. In particular, avoidance of over application, avoidance of application to saturated soils and application when plants are ready to immediately take up fertilizer can minimize excess nitrogen applications that result in elevated levels of NO3—N in the soil profile. NO3—N is readily converted to volatile N2O through the activities of soil microbes. The amount of N2O evolved from soil-applied nitrogen fertilizer is not trivial. In Canada alone, 9.2 megatonnes were estimated to be released (Art Jaques, P. EngChief—GHG Division, Environment, Canada) and the amounts released in the USA and other major agricultural countries would be expected to be much larger.
The present invention is directed to overcoming the deficiencies in the art.